The central oscillators of circadian clocks of organisms ranging from bacteria to humans synchronize biological activities with daily changes in sunlight and temperature, and have been shown to be important to health and survival. The central oscillator can be thought of as the heart of the circadian clock. What is the basis of the self -sustained rhythmicity, why are the periods ~24 hours, and why are the frequencies of circadian clocks much more resistant than the rates of most enzymes to changes in temperature? These questions are fundamental to all circadian central oscillators. Our long-term goal is to provide comprehensive answers to these questions for a central oscillator of a model system at biochemical and structural resolution. The central oscillator of the circadian clock in the cyanobacterium Synechococcus elongatus is comprised of only three proteins, KaiA, KaiB, and KaiC. A simple mixture consisting of the recombinant forms of these clock proteins and ATP in a test tube can reconstitute a self-sustained and temperature-compensated circadian rhythm of KaiC phosphorylation over several cycles. A single cycle has two halves: an ~12 hour autophosphorylation phase and an ~12 hour autodephosphorylation phase. Alternating interactions of KaiC with KaiA and KaiB, respectively, slowly swing KaiC between hyper- and hypophosphorylation over each day. This central oscillator-in-a-tube provides an outstanding opportunity to answer the questions posed above and to develop a biochemically and structurally detailed understanding of a biological timekeeper. Our specific aims are the following: 1. Determine how KaiA sets the ~12-hour autophosphorylation phase of the clock period. 2. Determine how KaiB mediates the ~12 hour autodephosphorylation phase of the clock period. 3. Determine whether autophosphorylation and autodephosphorylation of KaiC are cooperative processes. Here, we address biochemically and structurally the problems of how the long ~24 hour period of the circadian rhythm is established, how that period is buffered against changes in temperature, and how an oscillator reverses direction after 12 hours.